This project is focused on defining new mechanistic paradigms for Mycobacterium tuberculosis (Mtb) cell wall. biosynthesis and remodeling, which are essential for cell growth and division. Cell-wall biosynthesis is the target of well-known, critical anti-tuberculars, including isoniazid, cycloserine and ethambutol. We will concentrate on the pepfidoglycan (PG) layer of the cell wall, which serves as a meshwork for the structural integrity of the bacillus. Recent progress has identified proteins involved in PG homeostasis either enzymatically (PG hydrolases) or in regulatory roles (PknB, FhaA, and the lipid II flippase). These, along with other cell-wall biosynthetic enzymes, represent potential vulnerabilities that could be exploited for design of new TB drugs. We will take a multidisciplinary, multi-investigator approach enabled by our Core capabilities to address major questions about Mtb cell-wall biogenesis and its regulation. In Aim 1, we will define new molecular mechanisms of auto-inhibition of PG hydrolases to discover how their toxicity is mitigated. In Aim 2, we will structurally characterize complexes of active PG hydrolases. To uncover indirect cell-wall vulnerabilities, we also will determine the structures of complexes of regulatory factors that control PG biosynthesis in diverse environments. These studies will uncover for the first time activation mechanisms that control PG integrity. Aim 3 focuses on determining structures of small-molecule complexes of PG hydrolases and other cell-wall targets to define the basis for subrate- and inhibitor-binding specificty. By testtng fundamental hypotheses about cell-wall biosynthettc pathways and regulatory networks, this project sets the stage to develop potent, selective inhibitors of Mtb growth.